Whole genome sequencing characteristics of Chlamydia psittaci caprine AMK-16 strain, a promising killed whole cell veterinary vaccine candidate against chlamydia infection

Chlamydia psittaci is a primary zoonotic pathogen with a broad host range causing severe respiratory and reproductive system infection in animals and humans. To reduce the global burden of C. psittaci-associated diseases on animal welfare and health and to control the pathogen spread in husbandry, effective vaccines based on promising vaccine candidate(s) are required. Recently, the caprine C. psittaci AMK-16 strain (AMK-16) demonstrated a high level of protection (up to 80–100%) in outbred mice and pregnant rabbits immunized with these formaldehyde-inactivated bacteria against experimental chlamydial wild-type infection. This study investigated the molecular characteristics of AMK-16 by whole-genome sequencing followed by molecular typing, phylogenetic analysis and detection of main immunodominant protein(s) eliciting the immune response in mouse model. Similarly to other C. psittaci, AMK-16 harbored an extrachromosomal plasmid. The whole-genome phylogenetic analysis proved that AMK-16 strain belonging to ST28 clustered with only C. psittaci but not with Chlamydia abortus strains. However, AMK-16 possessed the insert which resulted from the recombination event as the additional single chromosome region of a 23,100 bp size with higher homology to C. abortus (98.38–99.94%) rather than to C. psittaci (92.06–92.55%). At least six of 16 CDSs were absent in AMK-16 plasticity zone and 41 CDSs in other loci compared with the reference C. psittaci 6BC strain. Two SNPs identified in the AMK-16 ompA sequence resulted in MOMP polymorphism followed by the formation of a novel genotype/subtype including three other C. psittaci strains else. AMK-16 MOMP provided marked specific cellular and humoral immune response in 100% of mice immunized with the inactivated AMK-16 bacteria. Both DnaK and GrpE encoded by the recombination region genes were less immunoreactive, inducing only a negligible T-cell murine immune response, while homologous antibodies could be detected in 50% and 30% of immunized mice, respectively. Thus, AMK-16 could be a promising vaccine candidate for the development of a killed whole cell vaccine against chlamydiosis in livestock.


Introduction
Chlamydia psittaci is Gram-negative, an obligate intracellular pathogen, one of the 14 characterized species of the family Chlamydiaceae [1][2][3].C. psittaci is globally recognized as the causative agent of a number of infectious diseases in animals, humans, poultry, reptiles, wild and decorative pet birds, especially parrots [1][2][3][4][5].Initially, these bacteria were associated with only avian psittacosis [4][5][6].However, since then it has been clearly proven that C. psittaci is also responsible for airway inflammation and reproductive disorders in cattle, small ruminants, domestic animals and humans [7][8][9][10][11][12][13]. C. psittaci (ovis), which is now re-classified to Chlamydia abortus, is responsible for abortion, stillbirth or fetal loss in ruminants worldwide.Small ruminants, sheep, and goats infected with this pathogen, may develop chlamydiosis, which is also known as ovine enzootic abortion (OEA) or enzootic abortion of ewes (EAE) [14].OEA is a common life-threatening infection in small ruminants whose outbreaks may cause global financial losses to the world animal husbandry industry.Therefore, each case of the disease demands an obligatory notification to the World Organization for Animal Health (WOAH) (https://www.woah.org/).
Both organisms, C. psittaci and C. abortus, are pathogenic to humans.As a rule, human infection is a result of inhaling the infected material derived from some zoonotic source, namely sick or infected livestock or birds.This can lead to animal and human respiratory diseases.Chlamydia infection in pregnant women may develop severe, sometimes life-threatening, outcomes in a mother herself, as well as result in stillbirth or miscarriage [15][16][17].
The organisms, C. psittaci and C. abortus, may establish in sensitive hosts either chlamydia monoinfection individually associated with each of these pathogens or simultaneous mixed infection caused by both chlamydia species [11,[18][19][20][21]. Cases of chlamydia infection in animals caused by chlamydia strains associated with recombination events in chromosomes were detected more often in C. psittaci than in C. abortus and were also reported for other Chlamydia spp.[22][23][24][25].
Virulence-attenuated or fully virulent strains of either C. psittaci or C. abortus have been actively tested for the development of either live or killed whole-cell vaccines (LWCV or KWCV, respectively) for prophylaxis of chlamydiosis in farm animals.However, recently a dramatic number of marked postvaccinal complications known as 'abortion storm' [26] has been registered in ewes after their immunization with the commercially available LWC vaccine based on the attenuated mutant C. abortus strain 1B.Additionally, this strain was proven to be transmitted from vaccinated to naïve unvaccinated animals, which resulted in the development of clinical features of a natural chlamydial wild-type infection [26][27][28][29].In fact, KWCVs intended for the prophylaxis of chlamydia in farm animals have been developed and successfully commercialized [30,31].This type of vaccines led to a decrease in chlamydia-associated abortion cases and absolutely abolished any vaccine-related complications such as chlamydia infection of the immunized sheep, transmission and uncontrolled spread of the relevant live vaccine strain from a vaccinee to naïve farm animals.Nevertheless, the efficacy of KWCVs remains questionable [32][33][34][35][36].This is a critical point in the development of effective chlamydia KWCVs for veterinary use, which highlighted the great importance of the evaluation of novel alternative vaccine candidate(s) based on wild virulent chlamydia strains.This could implicate the need to investigate the main genome characteristics of chlamydia strains recently derived from ruminants with chlamydiosis if those inactivated virulent isolates demonstrate a marked protective potency against experimental chlamydial infection.
Recently, it has been shown that immunization of small animal models (outbred mice and pregnant rabbits) with the formaldehyde-inactivated C. psittaci AMK-16 strain could induce a protection against model chlamydia wild-type infection.The immunized animals developed a detectable level of humoral response registered in the complement fixation test (CFT) with the use of the whole bacterial suspension as the antigen [37].However, neither the role of individual proteins in the elicited antibody response nor the cellular immune reactions were investigated in a well-characterized murine model widely used for immunogenicity research [30,31,[36][37][38][39][40][41][42][43][44][45][46][47].
Therefore, the main aim of this study was to sequence and investigate the main molecular characteristics of the C. psittaci AMK-16 strain genome in order to better understand: (i) the bacterial diversity of C. psittaci strains which currently cause chlamydia infection in small ruminants; and (ii) T-and B-cell immune response to the AMK-16 immunodominant proteins elicited by the immunization with the AMK-16 inactivated bacteria in a mouse model.

Bacterial strains
The Chlamydia strains used in this study are listed in S1 Table in S1 File.The C. psittaci AMK-16 was initially isolated in 2016 from the fetus of the aborted dairy goat under a local chlamydia outbreak in the private household in the Middle Volga Region of Russia.For wholegenome sequencing and immunological studies, the strain was routinely propagated in the yolk sac of chicken embryos following purification through Renografin density gradient centrifugation as previously described [6,22].The strain was kept either frozen at -70˚C or through lyophilization as reported [48,49].

DNA extraction and whole-genome sequencing
The total DNA was isolated from the AMK-16 lyophilized suspension using a commercial kit (DNA DNeasy Blood & Tissue Kit, Qiagen, Germany) according to manufacturer's instructions.The concentration of the extracted DNA was routinely measured using a spectrophotometer (Bio-Rad, USA).The DNA was stored at −20˚C until further use.
The annotation of this genome was performed by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/)[50].The complete genome of the AMK-16 strain was deposited at the NCBI GenBank (Acc.number for the chromosome is CP047319.1,and for the relevant plasmid, pAMK complete sequence-CP047320.1,S1 Construction of the phylogenetic tree for the C. psittaci and C. abortus strains was performed using the MEGA-7 software [51] by the maximum likelihood method.For this purpose, C. psittaci strains (n = 27) and C. abortus strains (n = 9) with the status of "complete assembly level" available in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 06 June 2023) were selected.Protein sequence-based genome comparison of AMK-16 and reference GR9 strains was performed using Proteome Comparison Service tool on The Bacterial and Viral Bioinformatics Resource Center (BV-BRC) platform [52].

Multi-locus sequence typing (MLST)
Multi-locus sequence typing (MLST) of the C. psittaci AMK-16 strain based on the concatenated fragments of seven housekeeping genes (gatA, oppA, hflX, gidA, enoA, hemN and fumC) was performed as previously described [22,54,55].The relevant sequence type (ST) for the AMK-16 was assigned by comparison with the allelic profiles for the Chlamydia strains available through the MLST online database [56,57] for Chlamydiales (http://pubmlst.org/chlamydiales/).The MLST-derived allele sequences of the AMK-16 were deposited in PubMLST (https://pubmlst.org/) with the access number/ID 4920.The minimum spanning tree based on the concatenated nucleotide sequences of seven house-keeping genes was constructed and visualized with the GrapeTree to reveal the phylogenetic relationships between the C. psittaci AMK-16 and other C. psittaci strains (n = 224) available in the PubMLST database (https://pubmlst.org/,accessed on 06 June 2023) (S2

ompA typing
The AMK-16 genotyping was applied to the full-length major outer membrane protein (ompA) gene sequencing derived from the relevant whole genome sequence available in the NCBI GenBank (Acc.number: CP047319.1, the locus tag GR632_02940).Phylogenetic analysis based on the ompA reference sequences derived from the ten C. psittaci classical genotypes (A-F, E/B, Mat116, M56 and WC) (S1 Table in S1 File) and the AMK-16 ompA sequence was performed with the use of the MEGA-7 software [51] by the maximum likelihood method.

Animal models and immunization
Mice.For immunization experiments the formalin-inactivated lyophilized C. psittaci AMK-16 bacterial suspension was diluted in sterile phosphate-buffered saline (PBS), and the total protein concentration of the resulting solution was determined by the Bradford assay.Six-to eight-week-old male outbred mice (n = 6) were immunized twice intraperitoneally at a 14-day interval with this C. psittaci AMK-16 bacterial suspension.The optimal immunization dose was determined in preliminary experiments and has been established as 20 mkg of protein per mouse.The inoculum was mixed 1:1 (v/v) with complete Freund's adjuvant (CFA) (Sigma, USA) for the first injection and incomplete Freund's adjuvant (IFA) (Sigma, USA) for the second immunization.
The control group of mice (n = 7) was inoculated with PBS mixed with CFA or IFA according to the same schedule.On the day 7 after the second injection all mice were sacrificed by blood withdrawal via cardiac puncture under the zolazepam-tiletamine plus xylazine terminal anesthesia, and spleens and cardiac blood were obtained for lymphocyte proliferation and serological assays, respectively.All animal procedures were carried out in strict accordance with the National Regulations.The animal study protocol was approved by the Institutional Review Board of the State Educational Institution of Higher Professional Education, Saratov Medical State University (IRB#00005197) and followed the rules stated in the Declaration of Helsinki.
Ewes.Naïve 6-8-month-old ewes (n = 8) were immunized i.m. three times with the inactivated bacteria of the C. psittaci homologous strain as described earlier [34].Sera were obtained from blood samples, lyophilized and stored for future investigations.

Evaluation of immune response elicited by the C. psittaci AMK-16 strain in animal models
Evaluation of humoral murine and ovine immune responses in immunoblotting.Sera collected from either mice or ewes immunized with the inactivated AMK-16 bacterial suspension were analyzed by immunoblot technique as was described previously [38], with the panel of the purified either recombinant or chemically isolated proteins including: (i) the MOMP derived from the AMK-16 (MOMP AMK-16) and the MOMP of the wild type C. psittaci GR9 reference strain (MOMP-GR9); (ii) the DnaK and the GrpE of the either AMK-16 or the C. psittaci GR9 (DnaK AMK-16, DnaK GR9, GrpE AMK-16 and GrpE GR9, respectively); (iii) the MOMP-containing C-antigen isolated from embryonated chicken eggs-derived (ECEs) suspension of chlamydia by a routine technique [6,22] after inoculation to ECEs of the C. psittaci AMK-16 (C-ag AMK-16).In addition, the whole-cell lysate of the C. psittaci AMK-16 suspension inactivated by heat exposure [6,22,37] was used.All recombinant antigens were ordered from AtaGenix Laboratories (China, http://en.atagenix.com).
Briefly, the antigens and bacterial lysate were resolved by electrophoresis in 12.5% SDS-PAGE [58], and the proteins were immediately transferred to a 0.22 μm pore size nitrocellulose membrane (Bio-Rad, USA).The membranes were blocked with 1% nonfat dried milk in PBS at room temperature for 1 hour with middle agitation.Then the membranes were washed twice in PBS and 0.05% Tween-20 (PBST) and incubated overnight at 4˚C with either murine or ovine sera diluted in 1% nonfat dried milk/PBST at the ratio 1:100 or 1:10-1:40, respectively.After four washings with PBST, the membranes were probed with either HRPconjugated goat anti-mouse or mouse anti-goat/sheep IgG (both Sigma, USA) at the dilution 1:25000 or 1:14000, respectively.Then, the washing step was repeated five times, and the reaction was developed with chromogenic substrate solution (3,3,5,5 0 -Tetramethylbenzidine (TMB) liquid substrate system for membranes, Sigma, USA).
Evaluation of cellular murine immune response in lymphocyte proliferation assay.Splenocytes collected from the mice immunized with the inactivated AMK-16 bacterial suspension were seeded at 1×10 5 cells/mL into 96-well cell culture plates in DMEM/F12 (Biolot, Russia) supplemented with 10% fetal bovine serum, FBS (Sigma, USA) and 0.1% gentamicin (Sigma, USA).The cells were stimulated with the C. psittaci recombinant antigens, namely MOMP-AMK-16, DnaK-AMK-16, GrpE-AMK-16, MOMP-GR9, DnaK-GR9, and GrpE-GR9 (all in the dose of 10 μg/ml).In separate experiments, C-antigen was also used in similar concentration, 10 μg of protein/ml.The plates were incubated at 37 ˚C in a 5% CO 2 atmosphere for 5 days.Unstimulated splenocytes and Concanavalin A from Canavalia ensiformis Type IV-S (ConA) (Sigma, USA) (2.5 μg/ml) were used as a negative and positive control, respectively.Lymphocyte proliferation was assessed with the use of Cell Proliferation ELISA BrdU chemiluminescent kit (Roche Applied Science, USA) according to the manufacturer's protocol.Stimulation indexes (SI) were calculated as the mean relative light units per second (rlu/s) of the antigen-stimulated cells divided by the rlu/s of the negative control.

Statistical analysis
The data from humoral and cellular immune response evaluation were analyzed and represented using the GraphPad Prism 6.01 (GraphPad software, San Diego, CA, USA).The Mann-Whitney test was used for detecting the differences between independent groups, and the Fisher's exact test was applied in the case of dichotomous variable, with the p-value <0.05 considered as significant for both statistical methods.

Brief whole genome characteristics of the C. psittaci AMK-16 strain
In this study, the initial whole genome sequencing which was performed using two platforms, NGS and MinION, successfully provided two separate contigs.The first of them corresponded to the C. psittaci bacterial circular chromosome of 1,152,497 bp and the other one was recognized as a single circular plasmid of 7,552 bp.As expected, similarly with other C. psittaci strains, the AMK-16 genome was relatively small and consisted of almost 1,000 coding sequences (CDSs), including all the three rRNAs types (5S, 16S and 23S), 38 tRNA genes, and 3 ncRNAs.In addition, 32 pseudo genes due to mainly frameshift mutations (n = 24 of 32) and the presence of stop codons in at least 9 of 32 DNA sequences (S3 Table in S1 File) were found.Importantly, the G+C content of the AMK-16 strain was 39.1%, which has been found to be identical to that for C. psittaci strains [23].The plasmid pAMK had eight coding sequences as in other known plasmid-bearing strains of Chlamydia spp.[23,59,60].
Phylogenetic analysis based on the whole genomes of all C. psittaci (n = 27) and C. abortus (n = 9) strains currently available in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 06 June 2023) and isolated from different sources demonstrated that the AMK-16 strain clustered only with C. psittaci but not with C. abortus strains (Fig 1).The AMK-16 formed a single branch only with another C. psittaci strain, also isolated from small ruminants [22], the C. psittaci Rostinovo-70 (Fig 1).The C. psittaci strain GR9, which was earlier (in 1960) isolated from a mallard in Germany [61], was identified as the closest homologue similarly with another study [62] and used as the reference one.Importantly, the C. psittaci strain 6BC, which was earlier recognized as the reference strain for the majority of C. psittaci representatives [63], formed another separate cluster of 15 relevant homologous strains.This cluster was phylogenetically distant from the other branch including, besides the AMK-16 & Rostinovo-70, and GR9, three other strains, the CPS-QD/LS, the WS/RT/E30 and the LS-QXC01, which, together with the AMK-16, formed a single cluster (Fig 1).The remaining C. psittaci strains formed three additional separate branches, each represented by a few (from 1 to 3) C. psittaci strains, such as: (i) the MN, the 01DC12 and the CP3; (ii) the NJ1, and (iii) the M56.The last two branches formed by two C. psittaci strains, the NJ1 and the M56, and isolated in the middle of the XX century, from either turkey or muskrat, were the most distant branches from the AMK-16-associated one (S1 Table in S1 File).These data could indicate the clear phylogenetic relation of the AMK-16 to C. psittaci species and divergence in relation to the most extensive cluster with the reference strain 6BC and to the minor ones as well.
Comparative analysis of the whole genomes of the C. psittaci AMK-16 versus the GR9 reference strain on the BV-BRC platform (https://www.bv-brc.org/)showed 100% homology in at least 865 CDSs, while 139 CDSs demonstrated a homology ranging from 73.4-99.9%(S4 Table in S1 File).Also, 41 CDSs were present in only the GR9 strain and absent in the AMK-16 strain.In fact, at least 35/41 CDSs were identified as uncharacterized hypothetical proteins.The remaining 6 CDSs might encode the following products: Phosphoglycolate phosphatase (EC 3.1.As expected, in BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi),this region, following the removal of other AMK-16 genome regions, showed a similar level of low homology (92.2%) to the corresponding areas of the whole genome sequences derived from the majority of the C. psittaci strains (n = 27) available in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/) independently from the isolation host (S5 Table in S1 File).A similarly low identity for this AMK-16 region (92.15% with 100% cover) was found with the analogous region in the C. psittaci GR9 reference strain.Only a single C. psittaci strain, the Rostinovo-70 [22], which had been earlier (more than 50 years ago) isolated from farm animals in Russia (S1 Table in S1 File), demonstrated the best match (100%) of the relevant region with that of the AMK-16 (S5 Table in S1 File).In fact, this AMK-16 region had a higher homology (in 98.38-99.94%range, with a total average of 98.57%) with the corresponding regions of all C. abortus strains (n = 11) (S5 Table in S1 File) which are currently also publicly available in the NCBI GenBank (https://  These data could indicate a possible gene recombination event(s) in the relevant region of the AMK-16 similarly with the Rostinovo-70 [22] and other Chlamydia spp.[23][24][25]54].

Molecular typing of the C. psittaci AMK-16 strain
MLST.MLST analysis using the concatenated fragments of the seven C. psittaci housekeeping genes (gatA, oppA, hflX, gidA, enoA, hemN and fumC) [22,23,54,55,64] of the AMK-16 strain and all other C. psittaci strains (n = 224) available in the PubMLST database (https:// pubmlst.org/)showed that the AMK-16 belonged to the ST28.It was the second largest cluster (n = 22), just after the ST24 (n = 123) (Fig 4a).Comparative analysis of the allele profiles of the AMK-16 and other representatives of ST28 and all the strains belonging to the ST24 demonstrated the difference in two variable alleles, gatA and fumC (S2 Table in S1 File).
The AMK-16 together with the Rostinovo-70 became the only strain of ST28 isolated from small ruminants (2/22, 9.1%).The majority of ST28 strains, including the GR9 and the WC/ RT/E30 organisms, were initially derived from bird specimens, ducks, poultry, ibis and mallard (16/22, 72.7%), while fewer were sourced from mammals, either human (3/22, 13.6%) or fox (1/22, 4.6%) samples (S2 Table in S1 File, Fig 4b).In contrast, more diversity was revealed in ST24 group, which involved the strains from birds (different psittacine birds) and mammals (exclusively horses and humans).The AMK-16 belonged to ST28 strains, which were mainly distributed in the European part of Eurasia, France, and Russia, while there were a number of   C. psittaci genotype G (following through the well-known classical A to F) or the subtype C1.However, the first suggestion may be more feasible, as the AMK-16 together with the other three strains formed a distinct branch which was clearly separated from the branch formed by two strains, the GR9 and the GD, and not overlapping either of them (Fig 1).In this case, the C. psittaci HB1043 could be further easily subtyped as the G1 genotype.In fact, the ompA sequence of the C. abortus CG1 was identical to the relevant gene of the AMK-16 and the Rostinovo-70, the whole genomes of which have been clearly proven to belong to C. psittaci spp.Thus, the ompA-based genotyping indicated that the CG1 strain could be reclassified as the C. psittaci CG1.However, neither the whole genome nor some other molecular targets of this strain are currently available to make the final decision on this matter.Similarly to the AMK-16 and the three other strains, the Rostinovo-70, the NB1043 and the CG1, the C. psittaci strains of genotype A, the 6BC, the 84/55 and the GIMS 2005:CpsCP1 clustered separately from the genotypes B, E and E/B strains as a single/individual genotype (Fig 5).The ompA sequence of the GIMS 2005:CpsCP1 was identical to that of the 84/55, indicating the same genotype A for both strains.However, both strains demonstrated certain discrimination from the 6BC, which probably identified them as the genotype A1.
Nevertheless, the AMK-16 together with the other three strains, the Rostinovo-70, the HB1043 and, probably the CG1 clearly formed individual genetic lineage related to ompA polymorphism.

Investigation of humoral and cellular immune responses induced by immunization with the inactivated C. psittaci AMK-16 strain in animal models
To evaluate the antibody response induced by the immunization of mice and ewes with the inactivated C. psittaci AMK-16 bacterial cells, an immunoblot with the panel of recombinant C. psittaci antigens, i.e., MOMPs, DnaK and GrpE, as well as with the MOMP-containing Cantigen and the C. psittaci AMK-16 bacterial lysate was performed.The comparison of nucleotide sequences for both DnaK and GrpE structural genes of the C. psittaci AMK-16 strain revealed a high (98.43-100%and 98.78-100%, respectively) level of identity with the relevant genes of C. abortus strains isolated from different animal hosts (S7 All the mice (100%) immunized with the inactivated C. psittaci AMK-16 developed antibodies strictly specific to the MOMP AMK-16 (Table 1).Notably, these sera reacted not only with the MOMP of the homologous C. psittaci AMK-16 strain, but also with the MOMP of the wild type C. psittaci GR9 reference strain, indicating that the humoral response induced by the AMK-16-based immunization could be addressed against the infection caused by the wild type C. psittaci strains.Similarly, clear immunodominant specific reaction with the band of 42 kDa corresponding to the MOMP antigen was detected in 100% of murine antisera when both Cantigen and the AMK-16 lysate itself were fractionated and used as sensitines for immunoblotting.No reaction with the MOMP-containing antigens was observed with the sera from the control unimmunized group.Thus, the MOMP-directed humoral response was highly specific (p<0.001)for the group of mice immunized with the inactivated C. psittaci AMK-16 bacterial suspension (Table 1).
Furthermore, slightly more than a half and less than a third of murine sera from the experimental group were positive to DnaK and GrpE proteins, respectively.This antibody response was likely non-specific, since the similar rate of positive responses was registered in the control group (p>0.05).Moreover, no significant differences have been found between the responses directed at these proteins of the C. psittaci homologous (the AMK-16) and wild-type (the GR9) strains (Table 1), despite a marked polymorphism in the AMK-16 genes encoding the AMK-16 DnaK and GrpE (S9 Table in S1 File).
As was expected, the dominant MOMP-directed humoral response was detected in the ewes vaccinated with the inactivated AMK-16 strain (S9 Table in S1 File).However, antibodies to both DnaK AMK-16 and DnaK GR9 were also registered in 87.5% and 62.5% of the immunized ewes, respectively.Only 25% of the ewes were seropositive to GrpE antigens.To assess the cellular antigen-specific murine immune response induced by the immunization with the inactivated C. psittaci AMK-16 bacterial suspension, a lymphocyte proliferation assay was performed.Middle lymphocyte proliferation was detected in the immunized group to both recombinant MOMP AMK-16 and MOMP GR9 stimuli (Fig 6).However, this response was significantly higher than those detected in the control unimmunized mice (p<0.05 and p<0.01, respectively).For the immunized group, the highest stimulation indexes, SI, were obtained to the C-antigen stimulus (p<0.001).This could indicate that the specific cellular immune response was directed not only at the MOMP, but also at the other components of the C-antigen, known as the ornithosis antigen, which is used for the conventional compliment fixation test in the investigation of EAE in ewes or chlamydia common antigen [39,65].No significant differences were observed in the lymphocyte proliferation activity induced by ConA between the groups, as well as in the proliferative response directed at both DnaK and GrpE proteins either of the homologous (the AMK-16) or the wild type (the GR9) C. psittaci strains, although a marked polymorphism of both AMK-16 antigens from the GR9 DnaK and the GR9 GrpE was detected (S8

Discussion
In this study, the AMK-16 strain that was recently isolated from the caprine aborted fetal tissue was investigated.This strain has demonstrated virulence in mouse model causing the death of 20, 70 and 100% of mice after subcutaneous, intraperitoneal, or intranasal infection, respectively.Moreover, the AMK-16 was virulent for guinea pigs [37].Simultaneously, the inactivated suspension of the AMK-16 strain showed a good level of protection against experimental chlamydia infection caused by C. psittaci wild strains sourced from cattle or small ruminants in an animal model.In fact, the immunization of white mice with the inactivated suspension of the AMK-16 by i.p. route could result in 2-4.6-fold and more survived animals (up to 85%) compared with the unimmunized naïve control group (no more than 20-35% of survivors) after i.p. administration with either bovine or ovine, or porcine, or homologous caprine virulent wild C. psittaci strains [37].Moreover, all 100% pregnant rabbits treated with the AMK-16 suspension following model infection with virulent C. psittaci wild strains, produced only alive offspring [37].The presence of the AMK-16 specific serum antibodies detectable in the CFT of 100% of vaccinated animals certainly correlated with the protective immune response induced by the immunization with the AMK-16 [37].However, the role of individual proteins in the elicited antibody or cellular immune responses has not been elucidated.Therefore, it was important to investigate the main molecular and immunological characteristics of this strain demonstrating its basic specific activities as a promising vaccine candidate for KWCV through whole genome sequencing followed by phylogenetic analysis, molecular typing, and comparison with the genomes of both C. psittaci and C. abortus strains available, and study of the immunodominant antigen target(s) of the AMK-16.
It is known that all Chlamydia possess a chromosome of the size around 1 Mbp which may be supplemented with an extrachromosomal circular replicon or cryptic plasmid of around 7.0-7.5 kbp in approximately 50-60% of isolated strains [23,59,60,[66][67][68][69].The AMK-16 whole genome sequenced had a chromosome of about 1.2 Mbp and the cryptic plasmid of about 7.5 kbp (S3 Table in S1 File), which corresponded to typical chlamydia representative genomes [3, 22-24, 30, 59-63, 70].Phylogenetic analysis based on the whole genome sequences of the AMK-16, C. psittaci and C. abortus strains available clear clustered the AMK-16 with only C. psittaci representatives (Fig 1).Furthermore, the AMK-16 had the GC content typical for C. psittaci [23] and belonged to ST28 which includes a number of C. psittaci strains (Fig 4a).Overall, this clustering was in agreement with the MLST clustering reported earlier [54,62,64,70].However, the AMK-16 became the second strain after the Rostinovo-70 grouped with the C. psittaci group II, which had initially included mainly the duck strains [54,64].Among them there was the C. psittaci GR9, the whole genome of which was found to be phylogenetically the closest homologue to the AMK-16 (Fig 1) similarly to the Rostinovo-70 as we reported earlier [22], which was then confirmed by recent studies from research groups [62,64,70].Comparative analysis of the whole genomes of these two strains resulted in the detection of a possible recombination event in the AMK-16 chromosome region with the size of 23.1 kbp coding 20 CDSs (Fig 2).It was not surprising, because the cases of chlamydia infection in farm animals caused by Chlamydia strains with chromosomes affected by recombinations have been reported [22][23][24][25]54].Importantly, those recombination events have been identified in C. psittaci in contrast to the more stable C. abortus with either little or no homologous recombinations associated with subsequent genome modification [24].As expected, this AMK-16 region was actually identical to that in C. abortus demonstrating a marked difference from those in C. psittaci representatives (S4, S5 Tables in S1 File).Recombination analysis based on the MaxChi method [53] strictly confirmed the phylogenetic position of this AMK-16 region in only the C. abortus-based cluster but not in the C. psittaci-related group (Fig 3).However, the comparison of the whole genomes of only the GR9 and the AMK-16 strains provided no marked difference in the PZ.That was revealed only when the 6BC and other C. psittaci strains, those phylogenetically more distant from the AMK-16 than the GR9 were compared (Fig 1, S6 Table in S1 File).In fact, at least six genes were lost in this region of the AMK-16.It is not clear whether or not some of them could be involved into the protective immune response conferring by the immunization of animal models with the AMK-16.These findings agree with the recent research, in which PZ has been carefully investigated in a number of C. psittaci strains [62].
A panel of recombinant or chemically derived proteins of the AMK-16 and the reference GR9 strains was used to evaluate both humoral and cellular immune responses elicited by the inactivated C. psittaci AMK-16 strain.The panel included the well-known immunodominant chlamydia antigen MOMP, MOMP-containing C-antigen and two proteins, Chaperone protein DnaK and Heat shock protein GrpE, encoded within the potential recombinational region of the C. psittaci AMK-16 strain.DnaK and GrpE are the stress response proteins, whose immunogenic and potentially protective properties were recently shown in different bacterial models [46,47,71,72].Both proteins demonstrated a certain variability in the AMK-16 compared with the GR9 (S2, S3 Figs in S1 File), while having almost identical amino acid lengths, 658/659 (DnaK) and 191/191 (GrpE), respectively (S4 Table in S1 File).For this reason, it was intriguing whether DnaK and GrpE could participate in the specific immune response induced by the immunization with the inactivated C. psittaci AMK-16 bacterial cells.In fact, at least one of these proteins, DnaK, clearly participated in the pathogen-host interaction following recognition by the host immune system of animal models, such as mice and ewes.Thus, DnaK could certainly support only the humoral host immune response in half of the mice immunized with the inactivated AMK-16 bacteria cells as the experimental model chlamydial KWCV (Table 1).In contrast, this protein showed better immunoreactivity in the target animal species, ewes, the sera of the majority of which tested positive for the presence of anti-DnaK antibodies (S7 Both humoral and cellular responses elicited by immunization with the inactivated C. psittaci AMK-16 strain were strictly MOMP-specific.Notably, these responses were targeted not only at the MOMP of the homologous C. psittaci AMK-16 strain, but also at the MOMP of wild type C. psittaci GR9.These findings are in good accordance with previous reports which state that the immune response induced by immunization with the inactivated C. psittaci AMK-16 could be effective against the infection caused by the wild type C. psittaci strains of different animal sources.Further on, the recombinant MOMP derived from the AMK-16 induced more marked T-cell proliferative response than that from the GR9 strain (Fig 6), although it was not statistically significant in a mouse model (p<0.05).In any case, this finding could indicate the importance for the protective host immune response of two non-synonymous substitutions in the nucleotide sequence of the ompA gene encoded MOMP in the C. psittaci strains with the novel genotype G (S1 Fig in S1 File), which were already detected in some countries, such as Russia and China.Importantly, this protein, MOMP, possessing both B-and T-cell immunoreactive epitopes, demonstrated high immunogenic activity.Therefore, nowadays natural and recombinant MOMP has been recognized as the most promising antigen for chlamydia vaccine development which completed the human clinical trial of Phase I [40][41][42][43][44][73][74][75].The data obtained in the current study could indicate that: (i) MOMP and DnaK were the two immunodominant antigens eliciting the host immune response in both laboratory and target animals after their immunization with the inactivated AMK-16 bacteria; (ii) the AMK-16 may be a promising vaccine candidate for development of veterinary KWCV against chlamydia infection, including EAO, in ruminants.In fact, the marked protective activity of the AMK-16 could be related to the specific genome characteristics, such as: (i) polymorphism in the ompA gene, resulting in the modification in MOMP amino acid compositions, which could lead to further evolution of C. psittaci strains and formation of the C. psittaci novel genotype variant that was differed from the genotype C; (ii) the presence in the C. psittaci chromosome a recombinant region highly homologous to C. abortus which encoded protein(s) with marked immunogenic potency.Some limitations of our study should be acknowledged.The main limitation was connected with the availability of a certainly small number of C. psittaci whole genomes with the status of "complete assembly level" which could be used to study the recombination event in these microorganisms.To avoid any inaccuracy in the phylogenetic relationships of the AMK-16 with other chlamydia, the extended collection of C. psittaci strains (n = 224) was carefully investigated using the PubMLST tools.Furthermore, the whole genomes of only a few C. psittaci strains, the Rostinovo-70 and the AMK-16 isolated in Russia, have been carefully investigated so far.Although both belonged to a single well-known clonal lineage (ST28) with other C. psittaci strains detected worldwide, they demonstrated a certain intraspecific genomic variation compared with others since the novel genotype G has been revealed.However, more isolates are needed to unravel the biodiversity of C. psittaci strains and the frequency of occurrence of strains with this genotype in different countries including Russia.Secondly, the present study was exclusively concentrated on the antigen-specific immune response elicited by the immunization with the inactivated AMK-16 bacteria, not their protective efficacy, since the high protective potential of the inactivated AMK-16 has been recently demonstrated in different laboratory models [37].Further, we did not measure the antibody titers in ELISA, only assessing antigen-antibody specific interaction in immunoblot assay to discover immunoreactive antigens of the C. psittaci AMK-16 strain, including those encoded within the recombinational insertion from C. abortus.The next limitation was that the panel of antigens used for serological and cellular response studies included only the AMK-16-and the GR9-derived antigens lacking those from other C. psittaci as well as from C. abortus strains, which would be advisable to test cross-reactivity with these two common pathogens of farm animals.Finally, although we assessed the proliferative cellular response induced by the inactivated AMK-16, we did not yet evaluate the antigen-specific cytokine profiles in both mice and target animals, although it was shown that protective immunity against chlamydia infection is strongly associated with the Th1-polarized immune response [76][77][78][79][80].

Conclusion
Our findings could result in an enhanced host immune response in animals immunized with the AMK-16.The KWCV based on the inactivated AMK-16 could be safe with no live vaccine-related complications in the form of clinical manifestations similar to chlamydia disease.Moreover, this vaccine may be universally protective for small ruminants and other livestock against C. psittaci strains sourced from different animal species, such as sheep, goats, pigs and cattle, as was reported recently [24,38].This vaccine, through the presence in the formulation of antigen composition from both C. psittaci and C. abortus, has a potency to protect animals infected with chlamydia belonging to either one of these pathogens or both of them.In fact, this characteristic needs to be carefully investigated in our future research.
3.18), Acetyl-coenzyme A carboxyl transferase alpha chain (EC 6.4.1.2),Transcription-repair coupling factor, CHLTR possible phosphoprotein, Uncharacterized protein CT719 and DNA mismatch repair protein MutL.Fig 2 highlighted a single chromosome region with a size of 23,100 b.p. located within 253 bp-276 bp from the replication point of the AMK-16 chromosome.This region included the 20 CDSs together with intergenic spaces which were arranged along the AMK-16 chromosome as a separate block.The relevant genes demonstrated a relatively low homology (from 74.7 to 98.8%, on average-91.9%)compared with those of the C. psittaci GR9 reference strain (S4

Fig 2 .
Fig 2. Circular representation of the genomes of the C. psittaci AMK-16 and the GR9 reference strain.The two circles inside (the purple diagram) show the distribution of the positions for protein-coding genes.The CDS identity is determined on a colorimetric scale, where purple/blue colors correspond to a higher percentage of identity than orange/red colored regions.The white areas represent the intergenic space in the chromosome sequence of the corresponding strain.The Figure was generated using the Proksee online tool (https://proksee.ca/,accessed on 06 June 2023).https://doi.org/10.1371/journal.pone.0293612.g002

Fig 3 .
Fig 3. Phylogenetic tree based on the C. psittaci AMK-16 recombination block and the relevant regions found in the whole genome sequences of the C. psittaci strains (n = 25) available in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/).This region of the C. psittaci AMK-16 is only clustered with the homologous regions of the C. abortus strains (n = 11) and the C. psittaci Rostinovo-70 genome [22], but not with the other representatives of C. psittaci indicated in S5Table in S1 File.https://doi.org/10.1371/journal.pone.0293612.g003

Fig 4 .
Fig 4. MLST Minimum spanning tree constructed using the GrapeTree algorithm based on the concatenated nucleotide sequences of 7 housekeeping genes (gatA, oppA, hflX, gidA, enoA, hemN and fumC) of the C. psittaci AMK-16 and all C. psittaci strains (n = 224) available in the PubMLST database (https://pubmlst.org/,accessed on 06 June 2023).The tree contains all sequence types (STs) currently identified in C. psittaci representatives.Each individual circle represents either: (a) a single sequence type (ST), or individual ST, sourced from specimens derived from: (b) different hosts, or (c) countries.The circle sizes are proportional to the number of strains.Links between circles represent the number of allelic mismatches between individual STs (S2 Table in S1 File).The AMK-16 belongs to ST28 presented as the light blue circle (a).https://doi.org/10.1371/journal.pone.0293612.g004

Fig 5 .
Fig 5. Phylogenetic tree based on the ompA gene sequences of the C. psittaci AMK-16 and the sequences of the ompA genes of the C. psittaci reference genotypes A-F, and C. abortus strains available in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/).The C. psittaci strain AMK-16 is formed a single branch with only the C. psittaci Rostinovo-70 strain (four strains forming with the AMK-16 individual branch colored as: red triangle, the AMK-16; green circles, the GR9 and GC or dark green squares, the CG1, the HB1043 and the Rostinovo-70).https://doi.org/10.1371/journal.pone.0293612.g005
Table in S1 File).

Table in S1
File).
Table in S1 File).The identity of DnaK and GrpE nucleotide sequences for the AMK-16 and the GR9 strains was 98.8% and 95.3%, respectively (S4 Table in S1 File).However, the relevant genes, dnaK and grpE, demonstrated a marked polymorphism in the AMK-16 strain versus the reference GR9 as 60 SNPs and 38 SNPs, respectively (S8 Table in S1 File, S2a, S3a Figs in S1 File).These nucleotide modifications resulted in 7 and 9 nonsynonymous substitutions in the AMK-16 relevant amino acid sequences of DnaK and GrpE (S8 Table in S1 File, S2b, S3b Figs in S1 File).

Table 1 . Study of the humoral immune response induced by the immunization of mice with the inactivated C. psittaci AMK-16 bacterial suspension in immunoblotting with the panel of C. psittaci antigens. C. psittaci antigens Number of sera with positive reaction in groups of mice P value 2 Immunized with the inactivated C. psittaci AMK-16 bacteria (n = 6) Control unimmunized (n = 7)
Table in S1 File, S2b, S3b Figs in S1 File).
Table in S1 File).GrpE clearly demonstrated negligible participation in the AMK-16-mediated antibody and cellular responses in only single animals in both mouse and ovine models (Fig 6).However, two other AMK-16 antigens, MOMP and MOMP-containing C-antigen, were identified as the AMK-16 immunodominant proteins, while demonstrating less polymorphism than both the AMK-16 DnaK and GrpE (S8 Table in S1 File, S2, S3 Figs in S1 File).